Neutrophil Extracellular Traps as Predictive Markers of Inflammation in Patients Undergoing Cardiac Surgery with Cardiopulmonary Bypass

Objectives: Cardiac surgery under cardiopulmonary bypass is associated with neutrophil activation and systemic inflammation (SIRS). In severe cases, SIRS can lead to organ dysfunction and even death. Neutrophil-derived proteases and reactive oxygen species contribute to tissue injury and endothelial dysfunction, predisposing patients to organ injury. In the past few years, the release of neutrophil extracellular traps (NETs) and their potent proinflammatory, cytotoxic and prothrombotic effects have gained much attention as risk factors for cardiovascular diseases and the development of postoperative complications. Here, we studied the role of NETs as inflammatory markers in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB).

Methods: Patients undergoing surgery with CPB were included in this prospective study. Blood samples were collected at the day of admission, before and after surgery, as well as postoperative at day 1, day 3, day 5, day 8, and day 10. Apoptosis rate of freshly isolated neutrophils was determined by propidium iodide staining of fragmented DNA and FACS analysis. In addition, toll-like receptor (TLR) 9 protein expression in neutrophils was examined by FACS. Levels of NETs were quantified in patients' plasma using Pico Green staining. The fluorescence intensity was quantified in a multiplate reader.

Results: Neutrophil apoptosis was markedly decreased immediately after surgery, suggesting neutrophil activation during systemic inflammation. Indeed, TLR9 expression levels in neutrophils were found to be significantly upregulated after surgery and remained high until day 10. Interestingly, TLR9 expression did not alter in patients with CPB< 100 minute when compared with the levels determined at the time of admission, whereas significant increase of TLR9 was found during the first 48 hour after surgery in patients with CPB>100 minute. Similar results were gained when NETs levels were quantified. Again, NETs levels significantly increased in patients with CPB>100 minute, but not in patients with CPB< 100 minute.

Conclusion: This study demonstrates that NETs might be used as predictive markers of CPB-mediated SIRS development. NETs levels directly correlate with neutrophil activation and TLR9 expression in these cells, suggesting a regulatory role of TLR9 for NETs formation.